| PurposeNumb gene is a newly discovered determinant of cell fate.It is closely related to the occurrence and development of various tumors.In previous experiments,our team demonstrated that the cell cycle of human prostate cancer PC-3 cells was blocked in G0/G1 phase through Hedgehog signaling pathway,after SaRNA was introduced into human castrated resistant prostate cancer PC-3 cells and with high expression.In this study,numb-overexpressed PC-3 cells were further used to form tumors on the surface of nude mice.HE staining,RT-q PCR,Western Blot and flow cytometry were used to verify the effect of high expression of Numb on the growth of prostate cancer and the cell cycle of human prostate cancer PC-3 cells through Hedgehog signaling pathway.Method 1.Thirty nude mice were randomly divided into three groups.Three groups of PC-3 cells in logarithmic phase were collected and cultured.Three groups of nude mice were subcutaneously injected with recombinant human PC-3 cells(recombinant PC-3 cells)with high expression of Numb gene as experimental group,PC-3 cells with blank transfection as control group and PC-3 cells without transfection as blank group.2.Fourteen days later,three groups of tumor tissues were taken for hematoxylin-eosin staining.3.Real-time quantitative PCR(RT-qPCR)was used to detect the mRNA expression of Numb,SHH,GLi and CyclinE in three groups of tumors.4.Western Blot method was used to detect the protein expression of Nmub,SHH,GLi and Cyclin E in tumorigenic tissues of nude mice.5.Flow cytometry was used to detect the cell cycle in tumorigenic tissues of three groups of nude mice.6.By comparing the results of hematoxylin-eosin staining,RT-q PCR,Western Blot and flow cytometry in three groups of tumors,the effects of up-regulation of Numb gene in human PC-3 cells on the expression of SHH,GLi and CyclinE were investigated.Result 1.After subcutaneous inoculation of PC-3 cells in nude mice for 2 days,a small white cuticle was observed at the site of inoculation,and gradually shrank.After 14 days,all nude mice inoculated with PC-3 cells successfully formed tumors.The growth rate of subcutaneous tumors in experimental group was lower than that in control group and blank group,and the tumors were smaller than that in control group and blank group.The tumorigenesis experiment showed that Numb gene inhibited the proliferation of CRPC cells in vivo and inhibited the tumorigenesis.2.hematoxylin-eosin staining showed that the proliferation rate of cells in the experimental group was lower than that in the control group and the blank group.3.RT-q PCR results showed that the transcription level of Numb gene in experimental group was significantly higher than that in control group and blank group(p < 0.05).There was no significant difference between control group and blank group(p > 0.05).The expression levels of SHh,Gli-1 and CyclinE genes in experimental group were significantly lower than that in control group and blank group(p < 0.05).4.Western Blot results showed that the expression of Numb protein in experimental group was significantly higher than that in control group and blank group,while the expression of SHh,Gli-1 and CyclinE protein in experimental group was significantly lower than that in control group and blank group(p < 0.05).There was no significant difference between control group and blank group.(p > 0.05).5.The results of flow cytometry showed that compared with the control group and the blank group,the cell cycle of transplanted tumors in nude mice was blocked in G0/G1 phase,the proportion of G0/G1 phase cells increased significantly,the proportion of S phase cells decreased significantly(p < 0.05),and the proportion of G2/M phase cells had no significant difference;there was no difference between the control group and the blank group(p > 0.05).ConclusionNumb-SaRNA can inhibit the growth of human prostate cancer PC-3 cells.The expression of SHH,GLi and CyclinE,which are important factors of Hedgehog signaling pathway,is decreased,and the cell cycle of PC-3 cells is blocked at G0/G1 phase.It is confirmed that up-regulation of Numb gene in CRPC cells by SaRNA can block the cell cycle of PC-3 cells at G0/G1 phase through Hedgehog signaling pathway.This conclusion is consistent at the cellular and animal levels. |
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